PURIFIED hCG

ABSTRACT

A process for the purification of recombinant human Chorionic Gonadotropin (hCG) from a sample of crude recombinant hCG in the supernatant of CHO cells comprises the combined use of ion-exchange chromatography and reverse phase HPLC. The ion-exchange chromatography is performed twice and the final use of a size exclusion chromatography allows the purification from any residual traces of contaminants. The specific bioactivity of the highly purified hCG obtained form the process is particularly high, amounting to about 25,000 IU/mg.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No. 10/204,630, filed Dec. 9, 2002, which is a 371 national stage of PCT/EP01/00665 filed Jan. 22, 2001, which claims priority to EP 00103690.4 filed Feb. 22, 2000. The entire contents of these applications are incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to a process for the purification of Chorionic Gonadotropin, in particular the purification of recombinant human Chorionic Gonadotropin (hCG) from a sample of crude recombinant hCG. The method comprises the use of ion-exchange chromatography and reverse phase HPLC.

Chorionic gonadotropin is a hormone produced by the placenta and traditionally obtained from the urine of pregnant women.

The hormone is a heterodimer consisting of non-covalently bound α and β subunits.

Its effects are predominantly those of the gonadotropin luteinising hormone.

Chorionic gonadotropin is given to women to induce ovulation after follicular development has been stimulated with follicle-stimulating hormone or human menopausal gonadotropins in the treatment of anovulatory infertility due to absent or low concentrations of gonadotropins. A single dose of 5000 to 10000 units is given by intramuscular injection to mimic the midcycle peak of luteinising hormone which normally stimulates ovulation. Chorionic gonadotropin is also given in conjunction with menotrophin and sometimes also clomiphene citrate as an adjunct to in vitro fertilization procedures and other assisted conception techniques involving superovulation and oocyte collection. In males it has been used in the treatment of prepubertal cryptorchidism. Regimens vary widely, but doses usually range from 500 to 4000 units three times weekly by intramuscular injection.

It is also given for male infertility associated with hypogonadotrophic hypogonadism. Again, there is considerable variation in the dosage regimen, and doses have varied from 500 to 4000 units two to three times weekly. An agent with follicle-stimulating activity such as menotrophin is often added to enable normal spermatogenesis. For oligospermia, doses of up to 3000 units of chorionic gonadotropin weekly with menotrophin or another follicle-stimulating preparation may be employed. In the treatment of delayed puberty associated with hypogonadism in males, a dose of 500 to 1500 units is given twice weekly; the dose should be titrated against plasma-testosterone concentration.

Different methods have been used to isolate and purify hCG from raw urine samples (Birken et al., Endocrinology, 133(3): 1390-7, 1993; Sakakibara et al., Chem. Pharm. Bull., 35(5): 1414-6, 1990; Donini et al., Acta Endocrinol., 73(1): 133-45, 1973). Recently, a different method of affinity chromatography, termed membrane filtration affinity chromatography, has been developed and applied to purify hCG from urine (Xu et al., Protein expression and purification, 16: 221-3, 1999). The method avoids the use of BrCN activated Sepharose as a solid-phase for the affinity chromatography column and represents a variation of the usual methods of purification of hCG by affinity chromatography from urine samples. Immunoactivity of the purified hCG according to this method is 8554 IU/mg.

Recombinant hCG has the advantage of being devoid of other gonadotropin hormones and contaminants of human origin and more specifically of those present in human urine. The crude preparation of recombinant hCG contains, however, all other proteins and contaminants of the cell used in its recombinant production and a method for achieving an absolute purity of recombinant Chorionic Gonadotropin is highly desirable.

SUMMARY OF THE INVENTION

We have now found that a crude preparation of hCG, deriving from a concentrated sample of a culture medium obtained after the recombinant process or from a crude concentrate of urine of pregnant women, can be purified such that the resulting hCG is practically free from proteins or other contaminants contained in the crude hCG preparation.

The purification process is based on the use of ion-exchange chromatography and reverse phase HPLC. The possible further use of a size exclusion column allows the removal of any residual traces of contaminants. Optimum results are obtained when at least two steps of ion-exchange chromatography are performed.

The process of the invention can be used for the purification of recombinant hCG from a crude preparation of the culture medium derived from the recombinant process. The r-hCG is obtained with a high degree of purity and high specific bioactivity (in the range of 23,000-28,000 IU/mg), practically free from Foetal Bovine Serum (FBS) proteins, if present in the culture medium, and from nucleic acids or other contaminants contained in the host cells used in the recombinant process.

The process of the invention can be used as well for the purification of urinary hCG, starting from a crude concentrate of urine of pregnant women, and for the purification of CG from other mammalian species including, for example, bovine, equine, porcine, ovine and monkey.

It is an object of the present invention to provide a process for purification of hCG from a sample comprising the use of ion-exchange chromatography and reverse phase HPLC.

The process comprises the steps of subjecting the sample to ion-exchange chromatography and subjecting the eluate to reverse phase HPLC. A further step of applying the eluate to a size exclusion column may additionally be carried out.

The two ion-exchange chromatography steps are preferably performed under different conditions in order to obtain optimum results from the purification process. A preferred embodiment of the process of the invention comprises the steps of:

(a) eluting the sample through a silica chromatography column;

(b) eluting through a DEAE SEPHAROSE (cross-linked agarose matrix with diethylaminoethyl weak anion exchanger) ion-exchange chromatography column;

(c) eluting through a CM-SEPHAROSE (cross-linked agarose matrix with carboxymethyl weak anion exchanger) ion-exchange chromatography column;

(d) eluting through a Silica C18 reverse phase HPLC column; and

(e) eluting through a SEPHACRYL (spherical allyl dextran and N,N′-methylenebisacrylamide) size exclusion chromatography column.

In a preferred embodiment of the invention, elution through the DEAE SEPHAROSE ion-exchange column is carried out in sodium phosphate buffer at about pH 7.5. Elution through the CM-SEPHAROSE (cross-linked agarose matrix with carboxymethyl weak anion exchanger) ion-exchange column is preferably carried out in sodium phosphate buffer at about pH 6. The reverse phase HPLC step (d) is preferably carried out with 2-propanol/Tris-phosphate buffer as mobile phase.

The CG of the present invention is preferably human CG and most preferably is recombinant hCG, deriving from the culture medium of CHO cells used in the recombinant process.

It is a further object of the present invention to provide a pharmaceutical composition comprising a therapeutically effective amount of purified recombinant hCG as prepared by the recombinant process as described above, together with suitable excipients. An example of a suitable excipient is sucrose, which aids in the stabilization of the lyophilized product. The pharmaceutical composition of recombinant hCG is particularity suitable for subcutaneous administration.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for the purification of hCG, in particular for the purification of recombinant hCG from a crude preparation of the culture medium of the recombinant process. The r-hCG is obtained with a high degree of purity and high specific bioactivity ( in the range of 23,000-28,000 IU/mg), practically free from Foetal Bovine Serum (FBS) proteins which are present in the culture medium and from nucleic acids or other contaminants contained in the host cells used in the recombinant process.

The invention is intended for use with biological materials, particularly crude mixtures containing hCG and other contaminating proteins referred to herein as starting material samples. The examples described in detail below use starting material samples containing r-hCG obtained from cell culture supernatant medium from a bioreactor.

Alternatively, the sample is crude concentrated urine from pregnant women.

The sample is constituted by freshly collecting cell culture supernatant medium perfused through a bioreactor over two days. Preferably the supernatant is clarified by filtration.

If necessary, the crude solution is concentrated and subjected to C4 silica chromatography to remove contaminants derived from the cell culture.

The semi-purified harvest, after ultrafiltration, is then subject to ion-exchange chromatography, which is preferably performed twice, and preferably under different conditions, and to reverse phase HPLC. A first DEAE SEPHAROSE ion-exchange step may be performed, essentially acting as an hCG “flow through” step, in which a large part of the non-hCG proteins and DNA are eliminated. A second ion-exchange step, preferably through a CM-SEPHAROSE column, acts as an hCG binding step, and removes residual DNA and host cell or medium protein contaminants. In a preferred embodiment this step is performed at about 5° C. eluting with sodium phosphate buffer at about pH 6.

Reverse phase chromatography on a Silica C18 column is effective in removing trace amounts of nucleic acids and cell culture derived contaminants. The column is preferably eluted with 2-propanol/Tris-phoshate buffer as mobile phase. The retentate solution is preferably then subjected to 10 kD cut-off ultrafiltration, concentrated and can be recovered with ammonium hydrogen carbonate pH 8. The concentrated product can then be applied to a size exclusion chromatography column on SEPHACRYL S200 HR. In this step, a separation based on molecular size is achieved eluting with ammonium hydrogen carbonate pH 8 to remove still possible trace amounts of cell culture derived contaminants, potential aggregates and free hCG sub-units. The eluate can then undergo a dialysis by ultrafiltration on membranes with 10 kD cut-off, preferably in sodium phosphate buffer, pH 7. After filtration, the purified hCG bulk is preferably stored in sterile bottles at low temperature.

EXAMPLE 1

Reagents:

Ammonia, analytical grade

Ammonium hydrogen carbonate, analytical grade (B.P.)

Di-sodium hydrogen phosphate, analytical grade

Absolute denatured Ethanol,

Phosphoric acid, analytical grade (Ph.Eur.)

2-propanol, analytical grade (Ph.Helv.)

Sodium chloride, analytical grade (Ph.Eur.)

Sodium di-hydrogen phosphate, analytical grade

Sodium hydroxide pellets, analytical grade (Ph.Eur.)

Trifluoroacetic acid (TFA), HPLC grade

Tris-(hydroxymethyl) aminomethane, analytical grade

Purification Process Summary Flow Diagram

Harvest material derived from the cell culture process is purified and concentrated by a series of five chromatographic steps.

The following flow diagram (Table 1) summarizes a preferred embodiment of the r-hCG purification process, outlining the chromatographic column resins and the principles of operation of each of the intermediate steps. TABLE 1 Flow diagram summarising the r-hCG purification process. Step I CULTURE MEDIUM FROM BIOREACTOR | C4 silica chromatography (Eluate contains r-hCG) | Ultrafiltration 10 kD cut-off (Retentate contains r-hCG) | CONCENTRATED CRUDE r-hCG HARVEST | Step II DEAE SEPHAROSE FF (Unbound fraction contains r-hCG) | Step III CM SEPHAROSE FF (Eluate contains r-hCG) | Step IV RP-HPLC ON SILICA C18 (Eluate contains r-hCG) | Ultrafiltration (10 kD) Step V SEPHACRYL S-200 HR (Eluate contains r-hCG) | r-hCG BULK SOLUTION

A detailed flow diagram and process description are provided below. The conditions given for the capture step (step I) are those which are normally applied when the crude material is of recombinant origin.

Step I (Capture Step)

In this step (Step I), a preliminary concentration is achieved and the buffer is changed to be of controlled composition. This step is initiated at room temperature (Silica C4 chromatography) and then continued at about +5° C. A preferred temperature range is 5±3. It is repeated individually for each harvest during the production cycle of the bioreactor.

(i) Clarification of Harvests

-   -   The freshly collected culture medium from the bioreactor is         usually first clarified by filtration.

(ii) Silica C4 Chromatography

-   -   After clarification, the harvests are loaded onto a C4 silica         chromatography column, which has been previously equilibrated in         sodium phosphate 25 mM, pH 7. A preferred pH range is from 6.6         to 7.7. The column is washed with sodium phosphate 25 mM until         the UV monitor signal returns to baseline. The product is then         eluted with 34.2% (w/w) 2-propanol in sodium phosphate 25 mM.

(iii) Ammonia Treatment

-   -   Ammonia is then added to the solution to reach a final         concentration of 1 M. This mixture is incubated for 6. hours.         Then the solution is 2 fold diluted with water, and the pH is         adjusted to 7.5 using phosphoric acid 85%. A preferred pH range         is 7.5±0.2.

(iv) Concentration and Dialysis

-   -   The 10 kD cut-off membranes stored in 0.05 M sodium hydroxide         between batches are rinsed with purified water until the pH         descends to approximately 8.     -   The product is concentrated and dialysed (by ultrafiltration on         the 10 kD membrane) to remove material having molecular weight         lower than 10 kD and to eliminate traces of 2-propanol and to         change the ammonia solution to sodium phosphate 40 mM pH 7.5. A         preferred pH range is 7.5±0.2     -   The final retentate is recovered from the membranes with sodium         phosphate 40 mM in order to achieve a target protein         concentration of 3 to 15 mg/ml.     -   The solution is then filtered and the resulting concentrate is         stored frozen at about −15° C.

Step II (Filtration and Ion Exchange on DEAE SEPHAROSE FF Chromatography)

This chromatography step is an r-hCG “flow-through” step in which a large part of the non r-hCG proteins and nucleic acids are eliminated. Whilst the filtration is carried out at room temperature, the chromatography stage where product passes through the column, is carried out in a cold room.

(i) Thawing and Pooling of the r-hCG Concentrated Crude Harvests

-   -   The frozen concentrates are thawed and pooled. A batch of         purified bulk r-hCG is processed from a pool of a variable         number of r-hCG crude concentrates produced from the same         working cell bank. The criteria for the number of r-hCG crude         concentrates pooled is based on the maximum protein binding         capacity of the next chromatographic step in the purification         process (4 mg total protein/mg of resin).

(ii) Clarification by Filtration

-   -   The r-hCG solution is preferably passed through a filter         apparatus and the filters washed with 40 mM sodium phosphate pH         7.5.     -   The filtered solution and washes are pooled.

(iii) Ion Exchange Chromatography on DEAE SEPHAROSE FF

-   -   The column, packed with a weakly charged anion-exchange resin,         diethyl amino ethane (DEAE) SEPHAROSE Fast Flow, is equilibrated         with 40 mM sodium phosphate (pH 7.5).     -   The r-hCG solution is loaded onto the column.     -   The column is fed with 40 mM sodium phosphate pH 7.5. The         chromatographic process is monitored by spectrophotometry at 280         nm.     -   The leading effluent is discarded until the peak starts eluting.         The unbound fraction containing the r-hCG is then collected.

Step III (CM SEPHAROSE FF Chromatography)

In this chromatographic step, a large part of the host cells contaminants are removed. The chromatographic step is carried out about +5° C. A preferred temperature range is 5±3.

(i) Dilution of the DEAE SEPHAROSE FF Eluate

-   -   Water for injection is added to the DEAE SEPHAROSE FF eluate and         the pH adjusted to 6 using phosphoric acid 85%. A preferred pH         range is 6±0.1.

(ii) Ion Exchange Chromatography on CM SEPHAROSE FF

-   -   The column, packed with a weakly charged cation-exchange resin,         Carboxymethyl (CM) SEPHAROSE Fast Flow, is equilibrated with 20         mM sodium phosphate buffer (pH 6). A preferred pH range is         6±0.1.     -   The r-hCG solution is loaded onto the column.     -   The column is washed with 20 mM sodium phosphate buffer pH 6.         The chromatographic process is monitored by spectrophotometry at         280 nm.     -   The product is eluted using 130 mM sodium phosphate buffer pH 6.         The leading effluent is discarded until the peak starts eluting.     -   The entire peak containing the r-hCG is collected. The product         can be optionally filtered at this stage to remove viral         contaminants.

Step IV (RP-HPLC on Silica C18)

This RP-HPLC chromatographic step is effective in removing trace amounts of cell culture contaminants, nucleic acid residues and endotoxins. It is followed by a 10 kD cut-off ultrafiltration and optional filtration.

(i) Preparation of the Aliquots

-   -   The pH of the aliquots is adjusted to 5 and 2-propanol is added         to a final concentration of 15% (v/v).

(ii) RP-HPLC Chromatography on Silica C18.

-   -   The column, packed with a Silica C18 resin is first equilibrated         in 15% (v/v) 2-propanol in Tris-phosphate 0.5 M buffer.     -   The first aliquot is loaded onto the column and the         chromatography is monitored by UV spectrophotometry.     -   The column is washed with the same equilibration buffer.     -   Elution of the r-hCG is subsequently performed with a linear         gradient of 2-propanol/Tris-phosphate 0.5 M buffer mobile phase         from 15% to 25% (v/v).     -   The r-hCG is fractionated when the corresponding peak is         detected by spectrophotometry (A₂₈₀). The fractions whose         absorbance is greater than 65% of the maximum peak height at the         ascending part and higher than 20% of the maximum peak height at         the descending part are pooled.     -   The four r-hCG containing pools are then pooled and diluted in         an equivalent volume of Water For Injection (WFI).     -   The product is concentrated and dialysed ( by ultrafiltration on         a 10 kD membrane) against WFI to remove material having         molecular weight lower than 10 kD and to eliminate 2-propanol.     -   The product is then dialysed by ultrafiltration against ammonium         hydrogen carbonate buffer 0.1 M, pH 8.     -   The resulting intermediate is stored at about +5° C. or frozen         if required. Preferred storage temperatures are 5±3° C. and         equal or below −15° C. respectively.

Step V (Size Exclusion Chromatography on SEPHACRYL S-200 HR)

This size exclusion chromatographic step is effective in removing trace amounts of cell culture derived contaminants, potential aggregates and/or free sub-units. It is followed by a 10 kD cut-off ultrafiltration. The Sephacryl S-200 HR and the 10 kD cut-off ultrafiltration steps are carried at about +5° C. A preferred temperature range is 5±3° C.

(i) Size Exclusion on Sephacryl S-200 HR.

-   -   The column packed with Sephacryl S-200 HR resin is equilibrated         with Ammonium hydrogen carbonate 0.5M (pH 8). A preferred pH         range is 8±0.2.     -   The r-hCG solution is loaded onto the column, and the elution         initiated using Ammonium hydrogen carbonate 0.5 M, pH 8. A         preferred pH range is 8±0.2.     -   The collection of the r-hCG fraction is initiated from the         beginning of the peak and lasts until the 50% mark of the         maximum peak height at the descending part of the peak is         reached.

(ii) 10 kD Cut-Off Ultrafiltration.

-   -   10 kD cut-off membranes stored in 0.05 M sodium hydroxide         between batches are rinsed with WFI until the pH descends to         approximately 8.     -   The product is concentrated and dialysed (by ultrafiltration)         against WFI.     -   The product is then dialysed (by ultrafiltration) against Sodium         phosphate buffer 0.01 M, pH 7, and the final protein         concentration adjusted to reach a target final concentration of         3.5 mg/ml.     -   The resulting r-hCG final bulk solution is preferably stored         frozen at about −15° C.

Chromatographic Resins

The following chromatographic resins may be employed in the purification process: Equivalent resins can also be employed.

Step I: Silica C4, 250 {umlaut over (Λ)}ngstrom—50 μm (Matrex®, Millipore)

Step II: DEAE SEPHAROSE FF (Pharmacia)

Step III: CM SEPHAROSE FF (Pharmacia)

Step IV: Silica C18, 300 angström—15-20 μm (Vydac)

Step V: SEPHACRYL S-200 HR (Pharmacia)

The current suppliers are:

Amersham Pharmacia Biotech, Millipore Corporation

Björkgatan 30 17 Cherry Hill Drive

S-751 84, Uppsala Danvers, Mass. 01923

Sweden USA

Vydac, The Separations Group,

17434 Mojave St.

Hesperia, Calif. 92345

USA

Results

Molecular Weight and Size

-   -   SDS-PAGE     -   The relative molecular weight of r-hCG obtained following the         purification method of the present invention has been determined         by SDS-PAGE against standard proteins of known molecular weight.     -   Coomassie brilliant blue staining after non-reducing SDS-PAGE         revealed a single broad band for the r-hCG heterodimer at         approximately molecular weight 70 kD (range 65-75 kD). The         identity of the band was confirmed by Western blotting.

Biological Activity

-   -   Biological Activity of different batches of r-hCG after         purification with the method of the present invention is         reported in Table 2. The protein concentration has been         determined by spectrophotometry at 276.5 nm, a=0.616.

The average specific activity of the r-hCG preparation is particularly high, amounting to about 25.000 IU/mg. TABLE 2 Specific bioactivity r-hCG batch (IU/mg) BCEA 9901 24427 BCEA 9902 26868 BCEA 9903 25636 BCEA 9904 27152 BCEA 9905 23729

Formulations

-   -   Both liquid and freeze dried formulations have been developed         with the highly purified recombinant hCG of the present         invention.     -   Liquid Formulation     -   Two liquid formulations at 10000 IU/ml were prepared in vials         DIN 2R using mannitol or sucrose as excipient and submitted to         stability tests at 50, 40, 25 and 4° C.

The composition is reported in tables 3 and 4. TABLE 3 Ingredients UNIT r-hCG IU/ml 10000 SUCROSE mg/ml 102.6 O. PHOSPHORIC ACID mg/ml 0.98 SODIUM q.s. to pH 7.0 HYDROXIDE Filling volume: 0.5 ml

TABLE 4 Ingredients UNIT r-hCG IU/ml 10000 MANNITOL mg/ml 54.6 O. PHOSPHORIC ACID mg/ml 0.98 SODIUM q.s. to pH 7.0 HYDROXIDE Filling volume: 0.5 ml

The results of the stability tests, carried out by Bioassay, SE/HPLC and RP-HPLC, showed that the mannitol formulation was more stable with respect to the sucrose formulation. Refrigerated storage conditions were preferably required to minimize the protein oxidation and free subunit formation.

Freeze Dried Formulation

A freeze dried formulation at 5000 IU strength was prepared in vials DIN 2R for stability tests at 50, 40, 25 and 4° C. using sucrose as excipient.

The composition is reported in table 5. TABLE 5 Ingredients UNIT r-hCG IU 5000 SUCROSE mg 30 O-PHOSPHORIC ACID mg 0.98 SODIUM q.s. to pH 7.0 HYDROXIDE

The results of the stability tests, carried out by Bioassay, SE/HPLC and RP-HPLC, showed that this freeze dried formulation was stable at 40 and 50° C. at least for 19 weeks.

The stability tests at 25 and 4° C. were performed up to 6 months indicating no degradation of the active substance. 

1. A recombinant human chorionic gonadotropin (hCG) having a specific bioactivity in the range of 23,000 to 28,000 IU/mg.
 2. A pharmaceutical composition comprising the recombinant hCG of claim 1, and suitable excipients.
 3. The pharmaceutical composition according to claim 2 for subcutaneous administration.
 4. A pharmaceutical composition according to claim 2, wherein the excipient is sucrose.
 5. The pharmaceutical composition according to claim 4 for subcutaneous administration.
 6. A pharmaceutical composition according to claim 4, which is a freeze-dried composition.
 7. The pharmaceutical composition according to claim 6 for subcutaneous administration.
 8. A pharmaceutical composition according to claim 2, wherein the excipient is mannitol.
 9. The pharmaceutical composition according to claim 8 for subcutaneous administration.
 10. A pharmaceutical composition according to claim 8, which is a liquid composition.
 11. The pharmaceutical composition according to claim 10 for subcutaneous administration. 